U.S. patent number 10,507,207 [Application Number 10/500,270] was granted by the patent office on 2019-12-17 for non-nucleosidic inhibitors of reverse transcriptase as antagonists of cell proliferation and inducers of cell differentiation.
This patent grant is currently assigned to ISTITUTO SUPERIORE DI SANITA. The grantee listed for this patent is Patrizia avia, Rodolfo Nello Lorenzini, Elisabetta Mattei, Clara Nervi, Gugliemo Palombini, Corrado Spadafora. Invention is credited to Patrizia avia, Rodolfo Nello Lorenzini, Elisabetta Mattei, Clara Nervi, Gugliemo Palombini, Corrado Spadafora.
View All Diagrams
United States Patent |
10,507,207 |
Spadafora , et al. |
December 17, 2019 |
Non-nucleosidic inhibitors of reverse transcriptase as antagonists
of cell proliferation and inducers of cell differentiation
Abstract
The invention refers to the use of Reverse Transcriptase (RT)
inhibitor compounds for the preparation of pharmaceutical
compositions to counteract the loss of cellular differentiation in
tumour and non tumour pathologies, said compound being able to bind
the hydrophobic pocket on the RT subunit p66. Particularly
preferred for such uses are the following compounds: nevirapine,
efavirenz, delavirdine, corresponding salts and/or pharmaceutically
acceptable derivatives thereof.
Inventors: |
Spadafora; Corrado (Rome,
IT), avia; Patrizia (Rome, IT), Mattei;
Elisabetta (Rome, IT), Palombini; Gugliemo (Rome,
IT), Lorenzini; Rodolfo Nello (Blera, IT),
Nervi; Clara (Rome, IT) |
Applicant: |
Name |
City |
State |
Country |
Type |
Spadafora; Corrado
avia; Patrizia
Mattei; Elisabetta
Palombini; Gugliemo
Lorenzini; Rodolfo Nello
Nervi; Clara |
Rome
Rome
Rome
Rome
Blera
Rome |
N/A
N/A
N/A
N/A
N/A
N/A |
IT
IT
IT
IT
IT
IT |
|
|
Assignee: |
ISTITUTO SUPERIORE DI SANITA
(Rome, IT)
|
Family
ID: |
26332794 |
Appl.
No.: |
10/500,270 |
Filed: |
December 23, 2002 |
PCT
Filed: |
December 23, 2002 |
PCT No.: |
PCT/EP02/14727 |
371(c)(1),(2),(4) Date: |
July 25, 2005 |
PCT
Pub. No.: |
WO03/055493 |
PCT
Pub. Date: |
July 10, 2003 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20060166970 A1 |
Jul 27, 2006 |
|
Foreign Application Priority Data
|
|
|
|
|
Dec 24, 2001 [IT] |
|
|
RM2001A0767 |
Aug 19, 2002 [IT] |
|
|
MI2002A1833 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
35/02 (20180101); A61P 35/00 (20180101); A61K
31/536 (20130101); A61K 31/496 (20130101); A61K
31/551 (20130101) |
Current International
Class: |
A61K
31/535 (20060101); A61K 31/536 (20060101); A61K
31/551 (20060101); A61K 31/496 (20060101) |
Field of
Search: |
;514/228.8,908 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Cortes et al. Cancer, (Mar. 1, 2002) vol. 94, No. 5, pp. 1492-1499.
cited by examiner .
Ghori A. et al. Telomerase inhibition as a potential new therapy
for colorectal cancer. Colorectal Disease. 2000;2(2):106-112. cited
by applicant .
Grimaudo S. et al. Selective induction of apoptosis in multidrug
resistant HL60R cells by the thiazolobenzoimidazole derivative
1-(2,6-difluorophenyl)-1H,3H-thiazolo [3,4-a] benzimidazole (TBZ).
Eur J Cancer. Oct. 1998;34(11):1756-63. cited by applicant .
Lam S. et al. Hypsin, a novel thermostable ribosome-inactivating
protein with antifungal and antiproliferative activities from
fruiting bodies of the edible mushroom Hypsizigus marmoreus.
Biochem Biophys Res Commun. Jul. 27, 2001;285(4):1071-5. cited by
applicant .
Modest G. et al. HIV and refractory anemia with excess blasts
(RAEB). Am J Hematol. Aug. 2002;70(4):318-9. cited by applicant
.
Murdaca G. et al. Complete remission of AIDS/Kaposi's sarcoma after
treatment with a combination of two nucleoside reverse
transcriptase inhibitors and one non-nucleoside reverse
transcriptase inhibitor. AIDS. Jan. 25, 2002;16(2):304-5. cited by
applicant .
Shaw A. et al. Kaposi's sarcoma regression following treatment with
a triple antiretroviral regimen containing nevirapine. Int J STD
AIDS. Jun. 1999;10(6):417-8. cited by applicant .
International Search Report for International Application No.
PCT/EP02/14727. cited by applicant.
|
Primary Examiner: Kantamneni; Shobha
Attorney, Agent or Firm: Nixon Peabody, LLP
Claims
The invention claimed is:
1. A method to treat a subject comprising the step of administering
to a subject identified as having a tumor selected from the group
consisting of a carcinoma, a fibro-sarcoma and an osteo-sarcoma an
effective amount of at least a compound selected from the group
consisting of, nevirapine, efavirenz, delavirdine, and
corresponding salts of nevirapine, efavirenz, and delavirdine to
exhibit an anti-tumor action.
2. The method according to claim 1 wherein the compound reconverts
differentiated cells of the tumor into phenotypically normal
cells.
3. The method according to claim 1 wherein said carcinomas are
selected from the group of teratocarcinoma, colon carcinoma, breast
carcinoma, adenocarcinoma, and hepatoma.
4. The method according to claim 1 wherein the compound is present
in a pharmaceutical composition that further comprises one or more
carriers and/or diluents and/or solvents and/or excipients suitable
for oral, intravenous, intramuscular or hypodermic injection
administration.
5. The method according to claim 1, wherein the compound is present
in a pharmaceutical composition in the form of a pill, suspension,
or solution.
Description
FIELD OF THE INVENTION
The present invention refers to non-nucleosidic inhibitors of
reverse transcriptase (RT) as antagonists of cell proliferation and
inducers of cell differentiation for therapeutical use in the
treatment and/or prevention of proliferative and differentiation
diseases such as cancer.
BACKGROUND OF THE INVENTION
Endogenous, non telomeric Reverse Transcriptase (RT) is an enzyme
encoded by two classes of abundant repeated elements in all
eukaryotic genomes: retrotransposons and endogenous retroviruses
(di Marzo Veronese F, Copeland T D, DeVico A L, Rahman R. Oroszlari
S, Gallo R C, Samgadharan M G Science (1986) 231,
1289-91--Characterization of highly immunogenic p66/p51 as the
reverse transcriptase of HTLV-III/LAV; Grob P M, Wu J C, Cohen K A,
Ingraham R H, Shih C K, Hargrave K D, McTague T L, Merluzzi V J
AIDS Res Hum Retroviruses (1992) 8, 145-52 Nonnucleoside inhibitors
of HIV-1 reverse transcriptase: nevirapine as a prototype drug).
Expression of RT-coding genes is generally repressed in terminally
differentiated non pathological, tissues--where it is detectable
only at a basal levels--yet is highly active in the mammalian
germline, embryonic tissues and tumor cells. The role played by RT
in such fundamental processes as cell growth and differentiation
remains to be clarified.
Nevirapine, Efavirenz and Rescriptor, also known under the
commercial names of VIRAMUNE.RTM., SUSTIVA.RTM. and RESCRIPTOR.RTM.
respectively, are known as non-nucleosidic inihibitors of RT and
are widely used in the therapy of AIDS as antiretroviral agents. In
particular nevirapine has the following empirical formula
C.sub.15H.sub.14N.sub.4O. In its pure state it is a crystalline
solid of molecular weight 266.302, with a melting point of
247-249.degree. C. and solubility of 0.1 mg/ml in water and 5.5
mg/ml in ethanol and can be prepared according to the indications
present in patent EP 429.987. Nevirapine
(5,11-dihydro-11-cyclopropyl-4-methyl-6H-dipyrido-[3,2-b:2',3'-e][1,4]dia-
zepin-6-one) is comprised in the group of compounds of the
5,11-dihydro-6H-dipyrido[3,2-b:2',3'-e][1,4]diazepines, which are
known as non-nucleosidic inihibitors of RT and used in the
prevention and treatment of HIV infections, as described in EP
429.987.
Efavirenz (M.W. 315.68)
(-)-6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4,-dihydro-2H-3,1-b-
enzoxazine-one [References herein incorporated by reference:
1.YOUNG, S. D.; BRITCHER, S. F.; TRAN, L. O.; PAYNE, L. S.; LUMMA,
W. C.; LYLE, T. A.; HUFF, J. R.; ANDERSON, P. S.; OLSEN, D. B.;
CARROLL, S. S.; PETTIBONE, D. J.; O'BRIEN, J. A.; BALL, R. G.;
BALANI, S. K.; LIN, J. H.; LONG, W. J.; BYRNES, V. W.;EMINI, E A.;
ET AL., L-743,726(DMP-266): A NOVEL, HIGHLY POTENT NONNUCLEOSIDE
INHIBITOR OF THE HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 REVERSE
TRANSCRIPTASE. ANTIMICROB AGENTS CHEMOTHER 39(12):2602-2605
(1995)]
Rescriptor (M.W. 552.68)
[1-(5-methanesulphonamido)-1H-indol-2-yl-carbonyl)-4-[3-(isopropylamino)--
2-pyridinyl]piperazine] (References herein incorporated by
reference: 1. ROMERO, D. L.; MORGE, R. A.; GENIN, M. J.; BILES, C.;
BUSSO, M.; RESNICK, L.; ALTHAUS, I. W.; REUSSER, F.; THOMAS, R. C.;
TARPLEY, W. G. BIS(HETEROARYL)PIPERZINE(BHAP) RT INHIBITORS:
STRUCTURE-ACTIVITY RELATIONSHIPS OF NOVEL SUBSTITUTED INDOLE
ANALOGUES AND THE IDENTIFICATION OF MONOMETHANESULFONATE(U-90152S).
J MED CHEM 36(10):1505-1508 (1993).
2. ROMERO, D. L.; OLMSTED, R. A.; POEL, T. J.; MORGE, R. A.; BILES,
C.; KEISER, B. J.; KOPTA, L. A.; FRIIS, J. M.; HOSLEY, J. D.;
STEFANSKI, K. J.; WISHKA, D. G.; EVANS, D. B.; MORRIS, J.; STEHLE,
R. G.; SHARMA, S. K.; YAGI, Y.; VOORM AN, R. L.; ADAMS, W. J.;
TARPLEY, W. G. TARGETING DELAVIRDINE/ATEVIRDINE RESISTANT HIV-1:
IDENTIFICATION OF (ALKYLAMINO)PIPERIDINE-CONTAINING
BIS(HETEROARYL)PIPERAZINES AS BROAD SPECTRUM HIV-1 REVERSE
TRANSCRIPTASE INHIBITORS. J MED CHEM 39(19):3769-3789 (1996).
Preliminary studies in our group have shown that both nevirapine
and efavirenz cause an early and effective developmental arrest in
early mouse embryos when added to cultures of embryos prepared in
in vitro fertilization (IVF) assays. That observation first
indicated that both drugs can potentiality inhibit cell
proliferation and prompted us to test their effect on tumor
cells.
SUMMARY OF THE INVENTION
The present invention is based on the finding that non-nucleosidic
RT inhibitors promote cell differentiation concomitant with
reduction of cell proliferation.
The term "inhibitor" as used herein refers to compounds that
interfere with the enzymatic activity through a direct binding with
RT molecules. More specifically, both nevirapine and efavirenz bind
the hydrophobic pocket on the RT subunit p66, which is localized
close to the catalytic site--the function of which is therefore
compromised. According to this definition, and within the scope of
the present invention, the commercially available compounds
mentioned above, i.e. VIRAMUNE (nevirapine), SUSTIVA (efavirenz)
and RESCRIPTOR (delavirdine), as well as other compounds capable of
interfering with RT activity, can be used in the therapy and/or
prevention of pathologies characterized by loss of cellular
differentiation and uncontrolled cell growth. Hence, the object of
the present invention is the use of non nucleoside compounds which
display RT inhibition activity according to the above mechanism,
which can be employed in preventive and/or curative therapy to
counteract the loss of differentiation in de-differentiating
pathologies and as antiproliferative drugs in tumour therapy. In
particular, RT inhibitors antagonizing the processes of cellular
proliferation and de-differentiation can be used in the therapy of
human tumors, in particular epithelial tumors, mesenchymal tumors
and tumors of the nervous system, including leukemias and solid
tumors such as teratocarcinomas, fibro- and osteo-sarcomas, colon
carcinoma, breast carcinoma, glioma and hepatoma.
Among non nucleoside RT inhibitors, the present invention
explicitly includes the use of commercially available compounds
which are currently used for the treatment of AIDS, which have
activity as non nucleoside RT inhibitors, including their relative
pharmaceutical forms. Among those, particularly preferred are:
Viramune.RTM. (nevirapine) (Boehringer), Sustiva.RTM. (efavirenz)
(Bristol-Myers Squibb) and Rescriptor.RTM. (delavirdine) (Agouron
Pharmaceuticals).
The above cited compounds, and nevirapine as a particular example,
in their commonly used and commercially available pharmaceutical
forms, are proposed as examples of compounds useful for the
preparation of pharmaceutical compositions to be employed in cases
in which cellular differentiation and/or proliferation must be
controlled, therefore with differentiating and anti-tumour
activity. The therapeutic effect of the molecules is to be placed
in relation to their RT inhibitory capacity.
A further object of the invention is the preventive or therapeutic
treatment of cell proliferation in mammals, in particular in
humans, with differentiating and anti-tumour actions.
Further objects will be evident from the detailed description of
the invention.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1: Reverse transcriptase in murine and human cell lines. FIG.
1A. Functional RT activity assay after incubation of MS2 RNA with
lysates from the following human (lanes 1-9) and murine (10-22)
cell types: lane 1, NB4 leukemia; lane 2, R4 leukemia; lane 3,
Kasumi-1 leukemia; lane 4, HL60 leukemia; lane 5, Saos-2
osteosarcoma; lane 6, MDA-231 breast carcinoma; lane 7, MCF7 breast
carcinoma; lane 8, U343 Mg glioma; lane 9, HT-29 colon carcinoma;
lane 10, NIH/3T3 embryo fibroblasts; lane 11, C2C7 myoblasts; lane
12, F9 teratocarcinoma; lane 13, L929 fibrosarcoma; lane 14,
control reaction with F9 lysate but no MS2 RNA; lane 15, buffer
only; lane 16, no mammalian cell lysate; lane 17, no MS2 RNA nor
cell lysate; lane 18, positive control reaction with commercial RT;
lane 19, no MS2-specific oligos; lanes 20-22: complete reaction
with F9 lysate pre-incubated with 1 (lane 20), 10 (lane 21) and 100
(lane 22) .mu.M nevirapine. Lanes M, DNA molecular weight markers;
lane 23, positive control reaction with commercial Rt. FIG. 1B.
Western analysis of RT proteins (upper panels) and .alpha.-tubulin
(lower panels) in WCE and nuclei. WCE were from: F9 (lane 1),
NIH/3T3 (lane 2), MCF7 (lane 3), MDA-231 (lane 4), NB4 (lane 5),
HL60 (lane 6) and ML2 blasts (lane 7). Nuclear extracts were from:
F9 (lane 1), MCF7 (lane 2), MDA-231 (lane 3), NIH 3T3 (lane 4),
HL60 (lane 5) and NB4 (lane 6).
FIG. 2: Nevirapine inhibits proliferation in murine and human cell
lines. Cells were cultured with (dashed line) and without (DMSO,
solid line) nevirapine. The proliferation rate is expressed as the
ratio of counted cells at the indicated times relative to the
initial number of seeded cells, taken as 1. Points represent the
mean value and bars the standard deviation from at least three
independent assays for all cell types.
FIG. 3A. Cell cycle analysis in control and nevirapine-exposed
cells. The distribution of cell cycle phases was determined by FACS
after 72 h from the beginning of nevirapine treatment. In the U343
glioma cell line, the cell cycle was separately analysed in the
diploid cell fraction (representing about 2/3 of all cells) and in
the remaining fraction (about 1/3) which develops aneuploidy. FIG.
3B. Western immunoblotting analysis of cyclin D1 (upper panel) in
extracts from the indicated cell types, cultured for 72 h with (+,
lanes 2, 4 and 6) or without (-, lanes 1, 3 and 5) nevirapine. The
filter was reprobed with anti-actin antibody to control equal
loading (lower panel).
FIG. 4: Differentiation of C2C7 myogenic cells after exposure to
nevirapine. C2C7 myoblasts pre-treated with DMSO (A, C) or
nevirapine (B, D, E) prior to culturing in differentiation medium
were stained with anti-MHC (FITC, green) and Hoechst 33258 to
visualize nuclei (in blue); merged pictures are shown. At 20.times.
magnification, myotubes in control cultures (FIG. 4A) are thinner
and less frequent than after nevirapine treatment (FIG. 4B). FIG.
4C, 60.times. magnification of myotubes in A. FIG. 4D, 60.times.
magnification of myotubes in B. FIG. 4E, 60.times. magnification of
MHC-positive myoblasts in the field in B.
FIG. 5: Morphological differentiation in RA- and nevirapine-exposed
F9 cells. F9 cultures exposed to DMSO (controls, FIG. 5a, FIG. 5b,
FIG. 5c, FIG. 5d), nevirapine (FIG. 5e, FIG. 5f, FIG. 5g, FIG. 5h)
or RA (FIG. 5i, FIG. 5j, FIG. 5k, FIG. 5l) were first examined in
vivo after 72 h of culture to record the morphological
reorganization (panels FIG. 5a, FIG. 5e, FIG. 5i, 60.times.
objective). After 96 h, samples were fixed and processed for IF of
collagen type IV (.alpha.1) chain (FIG. 5c, FIG. 5g, FIG. 5k) and
DAPI staining of nuclei (FIG. 5b, FIG. 5f, FIG. 5j); pictures are
merged in FIG. 5d, FIG. 5h and FIG. 5l (100.times. objective).
FIG. 6: Nevirapine treatment relieves the differentiation block in
AML blasts. Morphological differentiation, revealed by
Wright-Giemsa staining, in the promyelocytic cell line NB4; HL60
cells; and blasts from two AML patients (AML#1 and AML#2) after
treatment with 400 .mu.M nevirapine (Nev) for 5 days. NB4 cells
were also treated with 1 .mu.M RA, which induces granulocyte
differentiation. Untreated cells are shown as controls.
FIG. 7: Nevirapine induces variations in gene expression in F9
cells. Total RNA from F9 cells cultured with DMSO (controls),
nevirapine or RA was subjected to RT-PCR amplification with
oligonucleotides for the indicated genes, blotted and hybridized
with internal oligonucleotides. Representative panels are shown in
FIG. 7A. FIG. 7B: Quantitative variations in gene expression.
RT-PCR products hybridized with internal oligonucleotides were
quantified by densitometry. The signal ratio in nevirapine to
control cultures was normalized relative to that obtained for
.beta.-actin in the same experiment. Dark histograms represent the
mean value, and light histograms the standard deviation, from at
least three experiments for each gene.
FIG. 8: Efavirenz inhibits proliferation in murine cell lines (FIG.
8A 3T3 cell lines; FIG. 8B: F9 cells). Cells were cultured with
(dashed lines) and without (DMSO, solid line) efavirenz. The
proliferation rate is expressed as the ratio of counted cells at
the indicated times relative to the initial number of seeded cells,
taken as 1. Points represent the mean value from at least three
independent assays.
FIG. 9: Efavirenz inhibits proliferation in human cell lines. (FIG.
9A HeLa 1.sup.st cycle; FIG. 9B: HeLa 2.sup.nd cycle, FIG. 9C SAOS
1.sup.st cycle, FIG. 9D SAOS 2.sup.nd cycle). Cells were cultured
with (dashed line) and without (DMSO, solid line) efavirenz. The
proliferation rate is expressed as the ratio of counted cells at
the indicated times relative to the initial number of seeded cells,
taken as 1. Points represent the mean value from three independent
assays.
FIG. 10: Morris hepatoma growth in control (solid line) and
nevirapine-treated rats (broken line). The rate of tumor growth is
expressed as the volume (in cm.sup.3) over time (in days from the
time of inoculation).
FIG. 11: Tissue samples from nevirapine-treated (panels NEV) and
untreated sample (panels CTR) rats. In the upper panels (10.times.
objective), transformed areas in the NEV panel FIG. 11B are
remarkably reduced compared to the untreated (CTR) tissue (FIG.
11A). In the lower panels (40.times. objective), cells undergoing
apoptosis are clearly visible in the NEV (FIG. 11D) but not in the
CTR sample (FIG. 11C).
FIG. 12: Morris hepatoma growth in control (solid line) and two
efavirenz-treated rats (broken lines, EFV1 and EFV2). The rate of
tumor growth is expressed as the volume (in cm.sup.3) over time (in
days from the time of inoculation).
DETAILED DESCRIPTION OF THE INVENTION
We initially detected an endogenous RT activity, the enzyme
targeted by inhibitors, in a variety of murine and human tumor cell
line extracts using a PCR-based RT assay.
We then added 350-400 .mu.M nevirapine, or 10-20 .mu.M efavirenz,
for several days in cultures of murine progenitor cells (i.e., C2C7
myogenic precursor cells; NIH/3T3 embryo fibroblasts) and murine
and human tumorigenic cell lines (F9 teratocarcinoma; L929
fibrosarcoma; HT-29 colon carcinoma; MCF-7 breast carcinoma
expressing the estrogen receptor (ER+); MDA-231 breast carcinoma,
negative for ER expression (ER-); U343 Mg glioma and Saos-2
osteosarcoma). The results showed that RT inhibitors induce a
decrease in the rate of cell proliferation and promote cell
differentiation. Differentiation was also observed in acute myeloid
leukemia (AML) cell lines (NB4, HL60, Kasumi-1) and primary blasts
from two AML patients, as indicated by morphological, functional
and immunophenotypic assays.
RT-PCR analysis of mRNA extracted from F9 cells before and after
exposure to nevirapine depicted a substantial reprogramming of gene
expression in a set of genes which critically regulate the cell
cycle: cyclin D1 and D3 were down-regulated; conversely, their
antagonist p16 was up-regulated; to a lesser extent, the p27 kinase
inhibitor and the Rb-1 and Rb-2 retinoblastoma-related genes were
also down-regulated.
These results support the view that: a) an endogenous RT activity
is involved in tumorigenesis and b) RT inhibitors promote the
conversion of tumor phenotypes to normal phenotypes.
Nevirapine-induced differentiation was studied in greater detail in
multipotent F9 and myogenic C2C7 cell lines by following up the
appearance of specific differentiation markers that are not
expressed in progenitor cells, i.e. collagen IV .alpha.-chain in F9
cells and myosin in C2C7 cells, respectively. Moreover, studies at
the morphological (nucleo/cytoplasmic ratio and decreased
basophylia), functional (NBT assay) and immunophenotypic
(expression of lineage-specific surface antigens) levels indicate
that nevirapine treatment can rescue the differentiation block
present in human AML cell lines in primary tranformed blasts from
AML patients.
Based on these findings, we propose that the non nucleoside
compounds that show RT inhibition activity according to the above
mechanism be used in preventive and/or curative therapy as drugs to
counteract the loss of differentiation in de-differentiating
pathologies such as rhabdomyosarcoma, and as antiproliferative
drugs in tumour therapy, in particular epithelial tumors,
mesenchymal tumors and tumors of the nervous system, including
leukemias and solid tumors such as teratocarcinomas, fibro- and
osteo-sarcomas, colon carcinoma, breast carcinoma, glioma and
hepatoma.
Preferred are the compounds which are commercially available and
used for the treatment of AIDS which have activity as non
nucleoside RT inhibitors. Particularly preferred are: Viramune
(nevirapine) (Boehringer), Sustiva (efavirenz) (Bristol-Meyers
Squibb) and Rescriptor (delavirdine) (Agouron Pharmaceuticals).
The above cited compounds, and nevirapine as a particular example,
in their commonly used and commercially available pharmaceutical
forms, are proposed as examples of substances useful for the
preparation of pharmaceutical compositions to be employed in cases
in which differentiation must be controlled, at the same time
counteracting cellular proliferation, therefore with
differentiating and antitumour action. The therapeutic effect of
the molecules is to be placed in relation to their RT inhibitory
capacity.
The preventive or therapeutic treatment of cell proliferation
according to the invention can be performed in mammals, in
particular in humans.
The subjects in need can be treated with a therapeutically
effective amount of at least one compound that displays activity as
non nucleoside RT inhibitor and provides a therapeutic benefit to
the subject.
The non nucleoside compounds according to the invention can be used
in pharmaceutical compositions to prepare medicaments with
differentiating and antitumour action. The composition for the uses
described in the present invention may be obtained by mixing
together effective quantities of at least one active principle with
one or more physiologically acceptable carriers and/or diluents
and/or solvents and/or excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically, such as in form of pills, solutions,
suspensions. These pharmaceutical compositions may be manufactured
in a manner that is itself known, e.g., by means of conventional
mixing, dissolving, granulating, dragee-making, levigating,
emulsifying, encapsulating, entrapping or lyophilizing processes.
Proper formulation is dependent upon the route of administration
chosen. The pharmaceutical compositions may be administered orally,
or by intravenous intramuscular or hypodermic injection.
Uses, dosages and ways of administration are according to the
indications present in patent EP 429.987.
The doses and modalities of administration vary according to the
type and gravity of the affection.
This text will now proceed by describing some experimental examples
using the above mentioned molecules. However, it is to be stressed
that, owing to the different chemical structure of the compounds
that are preferred according to the present invention, it will be
understood that the invention is not limited to such molecules but
other compounds that display activity as RT inhibitor agents may be
applied as well.
The following examples are to be considered as illustrative and not
limiting of the scope of the present invention.
EXAMPLE 1
Endogenous RT Activity in Tumor Cells
The RT enzymatic activity, which is the target of the inhibitors
described herein, has been detected in all cell lines, of both
murine and human origin, that have been tested in this work using a
PCR-based assay. Results summarized in FIG. 1, A show that lysates
from all cell lines harbor a RT activity able to retrotranscribe in
vitro an exogenous RNA (MS2 phage RNA), generating an amplified
product of the expected size, i.e. 112 bp (lanes 1-13); the cDNA is
not synthesized if MS2 RNA (lane 14) or cell lysate (lane 16) are
omitted in the incubation mixture. Moreover, RT activity is
inhibited when cell lysate is pre-incubated with nevirapine in a
dose-dependent manner: the 112 bp-product is abolished after
incubation with 100 .mu.M (lane 22) but not with 1 (lane 20) or 10
.mu.M (lane 21). The presence of RT enzyme in tumor cells is
further confirmed by Western blot analysis. FIG. 1, B shows that
protein molecules are recognized by specific anti-RT polyclonal
antibody, both in whole cell (WCE) and in nuclear lysates.
These results-show that RT is present in tumor cells, both as a
protein and as an enzymatic activity, and that is inhibited by
nevirapine.
EXAMPLE 2
Incubation of Murine and Human Cell Cultures with Nevirapine:
Slow-Down of Cell of Cell Doublings
Murine C2C7 myogenic precursors, NIH/3T3 embryo fibroblasts, F9
teratocarcinoma and L929 fibrosarcoma cells, and human cells from
the following cell lines: Saos-2 osteosarcoma, HT-29 colon
carcinoma, MDA-231 breast carcinoma (ER-), MCF7 breast carcinoma
(ER+), U343 glioma, were cultured in DMEM containing 10-20% fetal
serum and 350 .mu.M nevirapine diluted from a stock solution (250
mM) in 100% DMSO. Cells were plated at a density of
2-5.times.10.sup.4 in 35 mm Petri dishes and exposed to nevirapine
5-6 hours later. Samples were withdrawn at specific times and cells
were counted in nevirapine-exposed and control (DMSO-exposed)
cultures. The results summarized in FIG. 2 show that exposure to
nevirapine (broken line) decreases the rate of proliferation in all
cell lines. Some cell type-specific differences were observed in
the response to the suppressive effect of the drug: F9
teratocarcinoma cells showed the highest response, with a 5-fold
reduction in the proliferation rate after 120 h of exposure. A
comparable effectiveness was depicted in HT-29 colon carcinoma
cells. Saos-2 osteosarcoma cells were the most slowly responsive
type, and only after 5 days of exposure did the growth rate begin
to decrease compared to control cultures.
EXAMPLE 3
Incubation of Cell Cultures with Nevirapine: Exit from Cell
Cycle
FACS analysis of nevirapine-exposed cell cultures depicted changes
in the cell cycle profile of several cell types: as shown in FIG.
3A, cells with a G0/G1 DNA content accumulated in NIH/3T3, HT-29,
MCF-7 and U343 Mg cultures after 72 h of nevirapine treatment,
whereas at these same times control samples displayed cell cycle
profiles typical of proliferating cultures. This was accompanied by
a substantial decrease in cyclin D1 levels in NIH/3T3, HT-29, MCF-7
(FIG. 3B), and U343 Mg glioma (data not shown) cultures exposed to
nevirapine compared to non-exposed controls.
EXAMPLE 4
Incubation of Cell Cultures with Nevirapine: Induction of
Differentiation
Nevirapine influences the process of cell differentiation as shown
using model cell systems capable of undergoing differentiation in
vitro with well characterized patterns. Murine C2C7 myogenic
satellite cells, which proliferate as mononucleated myoblasts can
be induced to differentiate upon growth factor withdrawal and form
multinucleated myotubes that express muscle-specific genes. In our
experiments C2C7 cells were cultured with or without nevirapine for
90 h (during which control cultures reached saturation density) and
then transferred to differentiation medium for 48 h. By light-field
microscopy scoring (n=300 cells from randomly selected fields), the
ratio between multinucleated myotubes (i.e., cells with more than
three nuclei) and mono/binucleated cells was about 1:1 in
nevirapine-treated compared to 1:2 in untreated samples. Cells
monolayers were further analyzed by immunofluorescence (IF) using a
polyclonal antibody to myosin heavy chain (MHC), a late marker of
muscle differentiation. In control cultures kept in differentiation
medium for 48 h, multinucleated MHC-containing myotubes were thin
and markedly smaller (FIG. 4C) than in cultures pre-treated is with
nevirapine (FIG. 4D). Isolated myoblasts were mostly MHC-negative
in control cultures (FIG. 4A), whereas myosin synthesis was already
activated in isolated myoblasts in nevirapine-treated plates (FIG.
4E).
Nevirapine also triggered differentiation in teratocarcinoma F9
cells. Untreated F9 cells have a rounded shape and tend to form
aggregates during growth (FIG. 5a). When exposed to retinoic acid
(RA), a well known promoter of differentiation, characteristic
signs of morphological differentiation become apparent after 72 h
(FIG. 5e), including a decreased tendency to form aggregates,
increased adhesiveness and reorganization of the cell surface with
the appearance of a differentiated morphology. Similar changes were
observed in nevirapine-exposed cultures (FIG. 5i). The
-morphological reorganization was accompanied by an increased
synthesis of collagen type IV(.alpha.1) chain (FIG. 5k), a marker
of differentiation induced in response to RA (FIG. 5g), synthesized
at low levels in control cultures (FIG. 5c). Together the results
in FIGS. 4 and 5 indicate that nevirapine triggers or facilitates
the onset of differentiation in two different cell types.
EXAMPLE 5
Nevirapine Treatment Induces Human Acute Myeloid Leukemia (AML)
Blast Differentiation
We analyzed the effect of nevirapine in the acute promyelocytic
NB4, expressing the t(15;17) oncoprotein PML/RAR, responsible for
the differentiation block present in these blasts, and the acute
myeloblastic leukemia HL60 cell lines, in comparison to their known
sensitivity to RA-induced granulocytic differentiation. After five
days of nevirapine treatment, the appearance of cells with a
myelomonocytic-like morphology was evident in both cell lines (FIG.
6), whereas cells with metamyelocyte-like morphology were induced
by 1 .mu.M RA, as reported (FIG. 6 and data not shown). We then
analysed the expression of the three surface antigens CD11b, CD14
and CD15, after four days of culture in the presence of RA or
nevirapine (Table 1). The induction of CD11b is associated with
granulocytic differentiation, CD14 is considered a monocytic
specific antigen and CD15 is a myelomonocytic antigen. Simultaneous
induction of CD14 and CD11b relates to monocytic differentiation.
Consistent with the induction of granulocytic differentiation, RA
treatment increased the levels of CD11b and CD15, but not CD14, in
both cell lines. In nevirapine-treated NB4 and HL60 cells, a strong
induction of expression of CD15, and, to a lesser degree, CD11b and
CD14 markers was detected. Thus, nevirapine treatment induces
expression of myelomonocytic differentiation markers, consistent
with the morphological studies.
Nevirapine treatment also increased the number of positive cells in
the NBT dye reduction assay by about 2.0-3.5 fold (Table 2).
The differentiating effect of nevirapine was further analysed in
myeloid leukemia cells poorly sensitive to RA-induced granulocytic
differentiation, such as the Kasumi-1 cell line expressing the
t(8;21) translocation product AML1/ETO, and in primary blasts from
two AML patients. Nevirapine treatment, though not actually
arresting proliferation, induced a modest yet constant accumulation
of cells in the G1 phase of the cell cycle (data not shown). In
addition, both Kasumi-1 cells and primary AML blasts responded to
nevirapine treatment by triggering differentiation, as revealed by
the induction of: (i) morphological changes related to
myelomonocytic differentiation (FIG. 6), consisting of chromatin
condensation with initial nuclear segmentation, decreased
nuclear/cytoplasmic ratio, decreased cytosolic basophilia and
appearance of a paranuclear Golgi region (specific granules were
evident in the case of AML#1); (ii) functional changes, indicated
by increased NBT positivity (Table 2); (iii) changes in the
expression of myeloid immunophenotypic markers CD11b, CD14 and CD15
(Table 1 and data not shown). The extent of induction of myeloid
differentiation features in primary AML blasts was comparable to
that detected in NB4 and HL60 cells. Insert Tab. 1 and Tab. 2
TABLE-US-00001 TABLE 1 Cytofluorimetric analysis of cell surface
markers in NB4 and HL60 cell lines after exposure for 4 days to RA
(1 .mu.M) or NEV (450 .mu.M): mean fluorescence intensity (AU) of
specific markers. Con- Con- Con- trol RA NEV trol RA NEV trol NEV
Markers NB4 cells HL60 cells AML#1 CD11b 7.2 53.7 10.1 12.1 15.3
16.6 5.6 5.9 CD14 18.3 19.1 23.1 34.3 23.7 45.3 8.1 8.8 CD15 106.5
421.7 196.3 122.5 609.7 1298.1 5.5 15.6
TABLE-US-00002 TABLE 2 Effect of RA or Nev on growth and
differentiation of NB4, HL60, Kasumi-1 cell lines and primary
leukemia blasts from two AML patients after exposure for 4 days.
Viable cells NBT+ Viable cells NBT+ N.sup.0 .times. 10.sup.5 (%)
N.sup.0 .times. 10.sup.5 (%) Treatment NB4 cells HL60 cells Control
2.6 .+-. 0.2 9.1 .+-. 0.5 9.2 .+-. 1.8 13.3 .+-. 0.8 RA (1 .mu.M)
1.4 .+-. 0.1 80.6 .+-. 4.5 6.5 .+-. 0.9 31.2 .+-. 1.4 Nev (350
.mu.M) 2.6 .+-. 0.4 37.2 .+-. 1.4 4.7 .+-. 1.1 37.8 .+-. 2.5 Viable
cells NBT+ Viable cells NBT+ Viable cells NBT N.sup.0 .times.
10.sup.5 (%) N.sup.0 .times. 10.sup.5 (%) N.sup.0 .times. 10.sup.5
(%) Treatment Kasumi-1 cells AML #1 AML #2 Control 3.3 .+-. 0.6
14.1 .+-. 0.5 0.9 .+-. 0.2 9.2 .+-. 0.4 1.8 .+-. 0.3 37.5 .+-. 4.9
Nev(350 .mu.M) 4.5 .+-. 0.9 31.2 .+-. 1.1 0.6 .+-. 0.1 19.5 .+-.
0.7 1.2 .+-. 0.1 68.1 .+-. 1.4
EXAMPLE 6
Altered Expression of Cell Cycle Regulatory Genes in
Nevirapine-Treated F9 Cells
To assess whether the ability of nevirapine to influence growth and
differentiation reflected changes in expression of specific genes,
RNA was extracted from control, nevirapine- and RA-exposed F9 cells
and subjected to semi-quantitative RT-PCR analysis. We examined a
set of genes encoding D-type cyclins; growth inhibitors; modulators
of apoptosis; and housekeeping proteins. Representative panels are
shown in FIG. 7A and results are quantified in FIG. 7B. Most
significant variations were recorded for the cyclin D1 gene, which
was down-regulated by 7-fold, whereas p16.sup.INK4a, encoding the
major antagonist of D-type cyclins, was up-regulated by nearly
8-fold, in nevirapine compared to non-exposed F9 cultures.
Up-regulation was also recorded for p27.sup.Kip1, which critically
modulates proliferation in response to cell shape and adhesion;
Rb-2/p130, which is associated with withdrawal from the
proliferative cycle; and Bcl-2, which can facilitate growth arrest
in some cell types. Cyclin D3 expression was down-regulated in
response to nevirapine. These changes are specific, because
expression of housekeeping genes (.beta.-actin, GAPDH), as well as
p53, was unaffected.
EXAMPLE 7
Incubation of Murine Cell Cultures with Efavirenz: Dose Effect
NIH/3T3 embryo fibroblasts and F9 teratocarcinoma cells were
cultured in DMEM containing 10-20% fetal serum and two
concentrations of efavirenz, i.e. 10 and 20 .mu.M, diluted from a
stock solution (10 mM) in 100% DMSO. Cells were plated at a density
of 2-5.times.10.sup.4 in 35 mm Petri dishes and exposed to
efavirenz 5-6 hours later. Samples were withdrawn at specific times
and cells were counted in efavirenz-exposed compared to parallel
non-exposed cell cultures. Results in FIG. 8 show that exposure to
efavirenz (broken line) decreases the rate of proliferation in both
cell lines in a dose-dependent manner as compared to non-exposed
cells (solid line).
EXAMPLE 8
Incubation of Human Cell Cultures with Efavirenz: Time
Dependency
The effect of efavirenz was further tested in human HeLa
adenocarcinoma and Saos-2 osteosarcoma cells. Both cell lines were
cultured in the conditions described above (Example 6). To
establish if prolonged exposure to the drug improved the
effectiveness of growth inhibition, both cell lines were exposed to
efavirenz in two subsequent cycles: during the 1.sup.st cycle cells
were continuously exposed to 20 .mu.M efavirenz for 5 days; cells
were then diluted, reseeded again and the 2.sup.nd cycle exposure
was initiated the following day with fresh drug. Results in FIG. 9
show that both HeLa (panels A) and Saos-2 (panels B) cell lines
were sensitive to efavirenz and underwent a significant reduction
in the rate of cell growth. The inhibitory effect was
time-dependent, since in both HeLa and Saos-2 cultures, the
1.sup.st cycle of exposure (5 days) yielded a 1.6- and 1.5-fold
reduction, respectively, whereas a 5- and 4-fold reduction was
obtained in the 2.sup.nd cycle (carried out until day 20 from the
onset of exposure).
EXAMPLE 9
Anti-Tumor Activity of Nevirapine in vivo: Treatment of Morris
3924A Rat Hepatoma
a) Rat Strain and Tumor Features
Rat strain: ACI/T inbred (about 180 gr)
Tumor: Morris 3924A hepatoma is a fast-growing tumor which develops
in inbred ACI/T animals. Three weeks after inoculation the tumor
size is about 10 cm.sup.3.
Preparation of tumor cells: hepatoma tumor cells are prepared from
the animal about two weeks after inoculation. The tumor is
surgically removed from the animal, separated from connective and
necrotic tissues and minced in small pieces. Tumor fragments are
then suspended in sterile physiological solution and inoculated in
the internal site of one thigh using a 20 ml-syringe with a large
size needle. Routinely 0.5 ml of cell suspension are injected. The
success rate of tumor implantation is nearly 99%.
Procedures: 3 rats were pre-treated with nevirapine by injecting
daily 0.2 ml/rat of nevirapine solution (stock solution=180 mg/2 ml
DMSO) for 11 days (18 mg/rat/day). On the eleventh day, hepatoma
cells were inoculated subcutaneously in pre-treated and in three
non-treated control rats.
Results: After 17 days from inoculation, control rats show typical
tumors of 2-4 cm.sup.3 whereas only one of the nevirapine
pre-treated animals shows a small nodule of a few millimeters in
the site of injection. The time curve in FIG. 10 shows the
differential rates of tumor growth in control (solid line) and
nevirapine-treated rats (broken line). The growth curve of controls
represents the average value of the three animals, whereas the
nevirapine curve refers to the growth rate of the single animal,
among those that were treated, which developed a small-size tumor.
Tumor tissues were surgically removed from both non-treated and
nevirapine-treated rats and submitted to histological analysis.
FIG. 11 shows that in the sample from nevirapine-treated rat
(panels NEV) the transformed areas are remarkably reduced, and
cells present clear apoptotic features, compared to untreated
sample (panels CTR).
In conclusion, pre-treatment with nevirapine effectively antagonize
the onset and the growth of an experimentally induced hepatoma in
rats (2/3) genetically predisposed to develop that specific tumor.
Two rats remained permanently healthy after tumor inoculation,
whereas in the third one a small-size tumor is detected. Under the
same conditions, untreated rats develop this fast-growing tumor (
3/3) and generally die within 27 days.
EXAMPLE 10
Anti-Tumor Activity of Nevirapine in vivo: Treatment of BALB/C Mice
Inoculated with Ascite Tumor
Procedure: 10 BALB/C mice were inoculated intraperitoneally (twice)
with ascite tumor cells by injecting 0.2 ml of ascite withdrawn
from an animal inoculated 8 days before. Treatment with nevirapine
started on the same day by injecting intraperitoneally 1
mg/mouse/day (stock solution 10 mg/ml in DMSO) in 5 of the 10 mice
inoculated with the tumor cells. Mice were treated continuously
with nevirapine for seven days.
Results: After 7-8 days, all control animals showed a swollen
abdomen containing 5-7 ml of ascite, as determined after the
animals were sacrificed. In contrast, three out of five (3/5)
nevirapine-treated animals remain healthy with no evidence of tumor
growth; the absence of tumor development was confirmed when 2 of
these animals were sacrificed 15 days after tumor inoculation and
the bodies were analyzed. The surviving treated animal remained
permanently healthy. It is worth recalling that mice inoculated
with ascite tumor survive only 12-14 days.
In conclusion, this experiment proves that nevirapine, injected at
the same time as tumor inoculation, blocks permanently the onset of
ascite tumor in three out of five mice. Under the same conditions,
the tumor developed in non-treated rats ( 5/5), all of which died
12-14 days after inoculation.
EXAMPLE 11
Anti-Tumor Activity of Efavirenz in vivo: Treatment of Morris 3924A
Rat Hevatoma
Procedures and Results: Morris hepatoma cells were inoculated in 4
rats (3924A strain). The same day, treatment with efavirenz was
initiated in two of them by injecting 1 mg/rat/day. FIG. 12 shows
the growth rate of the tumors in control (solid line) and
efavirenz-treated animals (broken line). The tumor grew rapidly in
control animals, which both died at the 27.sup.th day after
inoculation; in contrast only one of two treated animals developed
a tumor of markedly smaller size, whereas the other remained
totally healthy.
In conclusion, this experiment proves that treatment with
efavirenz, initiating the same time as tumor inoculation,
effectively antagonizes the onset and growth of Morris hepatoma. Of
two treated rats, one remained healthy several weeks after is tumor
inoculation, while the second one developed a markedly smaller
tumor. Under the same conditions, the tumor developed in
non-treated rats ( 2/2) and died at the 27.sup.th day.
* * * * *